CN111118151A - Human SMN1 and SMN2 gene copy number detection kit based on digital PCR method - Google Patents
Human SMN1 and SMN2 gene copy number detection kit based on digital PCR method Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to a human SMN1 and SMN2 gene copy number detection kit based on a digital PCR method, which comprises a DNA extraction reagent and PCR reaction liquid; the PCR reaction solution comprises primers for quantitative detection of human SMN1 and SMN2 genes, primers for detection of an internal reference RPP30 gene, human SMN1 and SMN2 gene detection probes and an internal reference RPP30 gene detection probe; the primer is adopted to amplify, so that the specificity, sensitivity and repeatability are high; the used probe improves the stability and affinity of DNA molecules in PCR reaction; the microdroplet digital PCR can directly detect the copy number of a target sequence, is greatly reduced by the influence of amplification efficiency, greatly improves the tolerance capability to PCR reaction inhibitors, has higher accuracy and repeatability in a detection mode, and can be applied to screening, clinical diagnosis, disease typing and the like of SMA carriers.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a human SMN1 and SMN2 gene copy number detection kit based on a digital PCR method.
Background
Spinal Muscular Atrophy (SMA) belongs to autosomal recessive neurodegenerative diseases and is caused by pure and deletion or mutation of SMN1 gene, and the SMN1 gene exists in chromosome 5q11-5q 13. SMA disease is a hereditary lethal disease second to vesicular fibrosis, the infant incidence is 1:6000 to 1:10000, and 1 heterozygote carrier exists in 40-60 people. SMA has been included in the list of rare diseases in the first group (No. 2018) established in association with 5 departments such as the national health council on 5/11/2018, and important attention is paid to these diseases on a national level.
Clinically SMA is divided into different sub-types depending on the severity of the disease and the age of the disease, type 0 being the most severe one, and patients require respiratory assistance from birth or shortly after birth, usually dying in as little as a few weeks. The most common type is type i (also known as Werdnig-Hoffmann disease), which typically occurs 6 months before, and is severely ill. These children cannot stand alone and will generally arrive at the end of life within 2 years. Type II is of medium severity type, the onset of disease is generally concentrated in 6-18 months, and the infant patient can stand independently. This type of infant has a shortened life expectancy, mainly due to the numerous complications of the respiratory system, most of which are adult despite a range of disabilities. Type iii (also known as Kugelberg-Welander disease) typically occurs around 3 years of age, and patients are able to walk and have a normal life expectancy. Finally, type iv is the mildest of the SMAs, is normally adult onset, has a normal life expectancy and a relatively better quality of life.
All SMA's have different degrees of clinical phenotype but the same genetic defect, homozygous deletion or point mutation of SMN1, resulting in the loss of SMN functional protein. Human possesses a unique copy of the SMN1 gene, SMN2, which has nearly the same coding sequence as the SMN1 gene. But there is a one nucleotide difference at the beginning of exon 7, resulting in a different alternative splicing of most transcripts of SMN 2. Normally, splicing of SMN1 results in the formation of a full-length mRNA and translation into a full-length SMN protein (294 amino acids, 32 kDa). The site CAGACAA at the 5' end of exon 7 in SMN1 represents an Exon Splicing Enhancer (ESE) sequence associated with the binding of splicing factors, facilitating the retention of exon 7 during splicing, thus forming the full-length SMN1 protein. SMN2 will become different from SMN1 if the C in the sequence becomes T, leading to the formation of exon splicing repressors (ESS), which facilitate exclusion of exon 7 during splicing by heterogeneous ribonucleoprotein a1 (hnRNP a 1) binding and result in the production of SMN2 Δ 7 mRNA and unstable SMN Δ 7 protein. The intronic splicing silencer N1 (ISS-N1) contains two hnRNP a1 binding sites and has a stronger exon 7 exclusion effect than the C-to-T conversion in the exon sequences. Nusinensesen and other antisense oligonucleotides (ASOs) target ISS-N1, thereby preventing binding to hnRNP a1 and promoting retention of exon 7 upon splicing and production of full-length SMN protein.
The study shows that the carrying frequency of SMN1 deletion mutation is different, and the carrying frequency of Asian is up to 2.4%. The SMN locus is located in an inverted repeat region on human chromosome 5 that contains a paralogous gene SMN2 of SMN 1. SMN2 was intact in all SMA patients. However, there are 0 and 4 copy number variations in SMN2 copy number in the general population. SMA patients carry at least 1 copy of SMN 2. 95% or more of SMA patients were caused by deletion of exon 7 of SMN 1. The number of copies of SMN2 correlated with the severity of the SMA phenotype, with more copies of SMN2 producing more SMN protein and less disease. The detection of the copy number of the SMN1/2 gene is an important means for SMA carrier screening and clinical confirmation and typing.
Traditional methods for detecting the copy number of exon 7 (ex7) of the SMN gene include polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and multiple ligation dependent probe amplification (MLPA). PCR-RFLP can only detect the homozygous deletion of the 7 th exon of SMN1, cannot detect carriers containing 1 copy, and cannot classify diseases. MLPA can detect homozygous deletion of exon 7, carriers containing 1 copy and normal people with 2 copies, but has complex operation and expensive reagents, and is not easy to popularize and apply clinically. There are some commercial kits for quantitatively detecting the copy number change of the SMN1 gene by a fluorescence quantitative method, but for the case of a light phenotype and distinguishing 1 copy from 2 copies, the fluorescence quantitative method firstly needs to make a standard curve, and cannot simultaneously detect SMN1 and SMN2, which leads to a significant rise in the reagent cost in clinical detection of a small amount of samples.
The digital PCR adopts an absolute quantitative mode, does not depend on a standard curve and a reference sample, and directly detects the copy number of a target sequence, and the principle is that a standard PCR reaction is distributed into a large number of tiny reactors, each reactor contains or does not contain one or more copies of target molecules (DNA templates) to realize single-molecule template PCR amplification, and after the PCR amplification, a biochip reader carries out fluorescence intensity detection on micro-droplets one by one. The intensity of the fluorescence signal of the positive microdroplets comprising the nucleic acid molecules is increased compared to the negative microdroplets. And finally, according to the principle of Poisson distribution, calculating the absolute copy number of the target nucleic acid molecule according to the proportion of the positive micro-droplets.
Disclosure of Invention
In order to solve the problems, the invention aims to disclose the field of molecular biology, in particular to a kit for detecting the copy number of human SMN1 and SMN2 genes based on a digital PCR method.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a human SMN1 and SMN2 gene copy number detection kit based on a digital PCR method, which is characterized by comprising a DNA extraction reagent and PCR reaction liquid; the PCR reaction solution comprises a primer for quantitative detection of human SMN1 and SMN2 genes, a primer for detection of an internal reference RPP30 gene, a human SMN1 and SMN2 gene detection probe and an internal reference RPP30 gene detection probe.
Preferably, the nucleotide sequences of the primers for quantitative detection of human SMN1 and SMN2 genes are shown as SEQ ID NO. 1 and SEQ ID NO. 2; the nucleotide sequence of the primer for detecting the internal reference RPP30 gene is shown as SEQ ID NO. 3 and SEQ ID NO. 4.
Preferably, the nucleotide sequences of the human SMN1 and SMN2 gene detection probes are shown as SEQ ID NO. 5; the nucleotide sequence of the internal reference RPP30 gene detection probe is shown in SEQ ID NO. 6.
Preferably, the 5 'end of the human SMN1 and SMN2 gene detection probe is marked with a fluorescent reporter group, the 3' end of the human SMN1 and SMN2 gene detection probe is marked with a non-fluorescent quenching group and is connected with an MGB modifying group, and one base of the human SMN1 and SMN2 gene detection probe is also subjected to locked nucleic acid modification; preferably, the fluorescent reporter group is FAM, TET, VIC, or HEX; the non-fluorescence quenching group is NFQ.
As a preferred mode, the quenching group adopted by the human SMN1 and SMN2 gene detection probe is a non-fluorescent quenching group, does not generate fluorescence per se, and can greatly reduce the intensity of a background signal; and the probe is a locked nucleic acid modified TaqMan MGB specific probe; the 7 th base of the mutant probe is modified by locked nucleic acid.
Preferably, the concentration of the primer for quantitative detection of the human SMN1 and SMN2 genes and the concentration of the primer for detection of the internal reference RPP30 gene are respectively 10-50 μm/L, and the more preferable concentration is 20 μm/L.
Preferably, the concentration of the human SMN1, the SMN2 gene detection probe and the internal reference RPP30 gene detection probe is 5-30 μm/L respectively, and the concentration is more preferably 10 μm/L.
Preferably, the PCR reaction solution further comprises a PCR Master Mix for microdroplet digital PCR.
Preferably, the DNA extraction reagent is QIAamp DNA Blood Mini Kit.
Preferably, the use of the kit comprises the following:
1) extracting DNA from a sample to be detected as a template; the concentration of the template is 1 ng/mul-to-50 ng/mul;
2) preparing PCR reaction liquid for detecting human SMN1 and SMN2 genes;
3) preparing a PCR specific amplification reaction system for droplet digital PCR detection of human SMN1 and SMN2 genes by using the DNA template extracted in the step 1);
4) generating microdroplets of the PCR specific amplification reaction system through a MicroDrop-100A sample preparation instrument;
5) performing digital PCR amplification; the specific amplification reaction procedure is as follows: 1 cycle at 50 ℃ for 2 minutes; 1 cycle at 95 ℃ for 10 min; 30 seconds of denaturation at 94 ℃, 60 seconds of annealing and extension at 58 ℃ for 45 cycles; 1 cycle at 98 ℃ for 10 min; keeping at 16 ℃;
6) and (3) calculating the copy number of the target molecule to be detected through software, specifically, detecting a fluorescence signal by using a MicroDrop-100B biochip analyzer, and calculating the copy number of the target molecule to be detected by using a MicroDrop ™ digital PCR (polymerase chain reaction) instrument matched data analysis software QuantDrop.
Preferably, the PCR-specific amplification reaction system of step 3) is formulated to include: PCR premix, SMN1/2 forward primer, SMN1/2 reverse primer, SMN1/2 probe, RPP30 forward primer, RPP30 reverse primer, RPP30 probe, ddH2O and a DNA template.
The invention has the beneficial effects that: the primers adopted by the invention are specific primers which are designed aiming at human SMN1 and SMN2 genes and are used for specifically amplifying the target region, and according to the design principle of the primers, the primers which are designed aiming at human SMN1 and SMN2 genes and can amplify the target region can be provided with a plurality of groups, and are not limited to the specific primers, but the primers of the invention have higher amplification specificity, sensitivity and repeatability, so the specific primers are selected from a plurality of primers.
According to the invention, a TaqMan MGB probe modified by Locked Nucleic Acid (LNA) is used, a quenching group of the probe adopts a Non-Fluorescent quenching group (Non-Fluorescent Quencher), and the self does not generate fluorescence, so that the intensity of a background signal can be greatly reduced; meanwhile, the probe is also connected with an MGB modifying group, so that the Tm value of the probe can be improved by about 10 ℃, the success rate of probe design is greatly improved, and the cost is reduced; in addition, the probe is modified by adopting Locked Nucleic Acid (LNA), so that the stability and affinity of DNA molecules in PCR reaction are improved, and the specificity and sensitivity are increased; the specific probe of the invention is adopted to obviously improve the specificity and the sensitivity.
The microdroplet digital PCR of the invention adopts an absolute quantitative mode, does not depend on a standard curve and a reference sample, directly detects the copy number of a target sequence, greatly reduces the influence of amplification efficiency on the reaction of the microdroplet digital PCR, greatly improves the tolerance to PCR reaction inhibitors, and has higher sensitivity, specificity, accuracy and repeatability.
The invention simultaneously and quantitatively detects the copy numbers of the SMN1 gene and the SMN2 gene of the EDTA anticoagulated whole blood sample, and can be applied to SMA carrier screening, clinical diagnosis, disease typing and the like.
Drawings
FIG. 1 is a two-dimensional graph of the present invention for detecting DNA of the SMN1 and SMN2 gene copy number HT1080 cell line by using digital PCR.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings:
the microdroplet digital PCR is an absolute quantitative technology of nucleic acid molecules, and the principle is as follows: after a standard PCR reaction system is subjected to microdroplet generation, each microdroplet contains or does not contain one or more copies of target molecules (DNA templates), single-molecule template PCR amplification is realized, after PCR circulation, microdroplets containing nucleic acid molecule templates can give fluorescence signals, microdroplets without the templates can not generate fluorescence signals, and finally, according to the Poisson distribution principle and the proportion of positive microdroplets, the concentration or copy number of the target molecules to be detected can be calculated by adopting a Microdrop algorithm digital PCR instrument matched data analysis software Quantdrop; the digital PCR can directly calculate the copy number of a target sequence, so that accurate absolute quantitative detection can be carried out without depending on a control sample and a standard curve; in addition, because the digital PCR only judges whether two amplification states exist or not when the result is judged, the intersection point of a fluorescence signal and a set threshold line does not need to be detected, and the identification of a Ct value is not relied on, the influence of the amplification efficiency on the reaction of the digital PCR is greatly reduced, and the tolerance capability on a PCR reaction inhibitor is greatly improved; the process of standard reaction system distribution in the digital PCR experiment can greatly reduce the background sequence concentration which has a competitive action with a target sequence, and the concentration or copy number of the target molecule to be detected is calculated.
The probe is a TaqMan MGB specific probe modified by Locked Nucleic Acid (LNA), the Locked Nucleic Acid (LNA) is a special bicyclic nucleotide derivative, the structure contains one or more 2' -O, 4' -C-methylene-D-ribofuranosyl nucleic acid monomers, the 2' -O site and the 4' -C site of ribose form an oxymethylene bridge-thiomethylene bridge or an aminomethylene bridge through different glycidyl actions and are connected into a ring, and the ring bridge locks the N configuration of a furanose C3' -endo form, so that the flexibility of a ribose structure is reduced, the stability of a partial structure of a phosphate skeleton is increased, the stability and affinity of DNA molecules in PCR reaction are improved, and the specificity and sensitivity are increased.
Minor groove binders (MGBs for short) are chemical groups derived from some antibiotic molecules, can be embedded into minor grooves in a DNA double-helix structure to form non-covalent binding, can generally improve the Tm value of oligonucleotide by about 10 ℃, can shorten the length of a primer or a probe, improve specificity and bring convenience to design; the MGB greatly improves the success rate of probe design and reduces the cost.
In the following examples, the MicroDrop-box digital PCR system is from Guangdong Yongno medical science and technology, Inc., and comprises a MicroDrop-100A sample preparation instrument and a MicroDrop-100B biochip analyzer, and the experimental methods used in the following examples are conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The first embodiment is as follows: human SMN1 and SMN2 gene copy number detection primer based on digital PCR method
In an embodiment of primers for quantitative detection of the SMN1 and SMN2 genes of the present invention, the primers for quantitative detection of the human SMN1 and SMN2 genes described in this embodiment are as follows:
a forward primer: TAACTTCCTTTATTTTCCTTACAGGG (shown as SEQ ID No. 1);
reverse primer: CATAATGCTGGCAGACTTACTCCT (shown in SEQ ID No. 2).
Designing a primer: searching a cds sequence of a human SMN gene from NCBI, and designing an upstream primer and a downstream primer which can amplify a segment containing exon 7 by using primer software; according to the principle of primer design, a plurality of pairs of primers are designed. A plurality of pairs of primers comprising the primers shown as SEQ ID NO. 1 and SEQ ID NO. 2 are synthesized, and 6 pairs of primers are tested by adopting a PCR technology, so that the primers can influence the length of an amplified fragment, and the sensitivity of the experimental scheme can be influenced by different amplified lengths. Also, the efficiency of amplification differs for different primers.
Researches show that the primers shown in SEQ ID NO. 1 and SEQ ID NO. 2 have higher amplification specificity and efficiency, and the sensitivity of detection by adopting the primers is higher.
An example of the primer for detecting the internal reference RPP30 gene of the present invention is as follows:
a forward primer: TCAGCATGGCGGTGTTTG (shown in SEQ ID No. 3);
reverse primer: CGCAGAGCCTTCAGGTCAG (shown in SEQ ID No. 4).
Designing a primer: searching a cds sequence of a human RPP30 gene from NCBI, and designing an upstream primer and a downstream primer by using primer software; according to the principle of primer design, a plurality of pairs of primers are designed. A plurality of pairs of primers comprising the primers shown as SEQ ID NO. 3 and SEQ ID NO. 4 are synthesized, and the 4 pairs of primers are tested by adopting a PCR technology, so that the primers can influence the length of an amplified fragment, and the sensitivity of the experimental scheme can be influenced by different amplified lengths. Also, the efficiency of amplification differs for different primers.
Researches show that the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 have higher amplification specificity and efficiency, and the sensitivity of detection by adopting the primers is higher.
Example two: human SMN1 and SMN2 gene copy number detection probe based on digital PCR method
The invention relates to an embodiment of SMN1 and SMN2 gene detection probes, which consist of human SMN1 and SMN2 gene detection probes (combined with a DNA template); the Probe is dd-SMN1-Probe 3: 5 '-FAM-TTGTCT + G AAACCCTGT-MGB-NFQ-3' ("+ N" indicates that the base is LNA modified) (as shown in SEQ ID NO: 5); namely: the 5 'end of the mutant probe is marked with FAM, the 3' end of the mutant probe is marked with a non-fluorescence quenching group NFQ and is also connected with an MGB modifying group, and the 7 th base of the probe is subjected to locked nucleic acid modification.
In one embodiment of the detection probe of the internal reference RPP30 gene, the probe consists of a detection probe (combined with a DNA template) of the internal reference RPP30 gene; the Probe is dd-RPP30-Probe 1: 5 '-FAM-AGATTTGGACCTGCGAGC-MGB-NFQ-3' (shown in SEQ ID NO: 6); namely: the 5 'end of the mutant probe is marked with FAM, the 3' end is marked with a non-fluorescence quenching group NFQ, and meanwhile, the mutant probe is also connected with an MGB modifying group.
The specific probe of the embodiment is matched with the specific primer of the embodiment 1 for detecting the copy number of the human SMN1 and SMN2 genes.
Example three: kit or PCR reaction solution for detecting copy numbers of human SMN1 and SMN2 genes
An example of the PCR reaction solution for detecting the copy number of SMN1 and SMN2 genes of the present invention is shown in table 1; wherein, the PCR premix is PCR premix Master Mix matched with a MicroDrop ™ digital PCR instrument of Guangdong Yongno medical science and technology Limited company; the forward and reverse primers are shown in example 1.
TABLE 1
In the experimental process, the PCR reaction solution of Table 1 was mixed with 2. mu.l of DNA template to obtain 20. mu.l of PCR specific amplification reaction system.
The Kit of this example comprises the PCR reaction solution of this example and a DNA extraction reagent, wherein the DNA extraction reagent is QIAamp DNA Blood Mini Kit.
Example four
In this embodiment, the PCR reaction solution/kit described in example 3 is used to detect exon 7 of SMN1 and SMN2 genes, and the specific detection method is as follows:
(1) extracting genome DNA: extracting normal peripheral Blood genomic DNA and HT1080 cell line DNA by using a QIAamp DNA Blood Mini Kit according to the Kit instruction, detecting the purity of the obtained genomic DNA by using NanoDrop 100, detecting the integrity of the DNA by gel electrophoresis, and quantifying by using a qubit4.0 fluorimeter, wherein the concentration range of the DNA is 20 ng/mu l-50 ng/mu l.
(2) The PCR reaction solution for detecting the copy number of the SMN1 and SMN2 genes by microdroplet digital PCR is prepared as follows: 10 μ L of PCR premix Master Mix, 0.5 μ L of 20 μm/L SMN1/2 forward primer, 0.5 μ L of 20 μm/L SMN1/2 reverse primer, 0.5 μ L of 20 μm/L RPP30 forward primer, 0.5 μ L of 20 μm/L RPP30 reverse primer, 0.6 μ L of 10 μm/L SMN1/2 probe, 0.6 μ L of 10 μm/L internal reference RPP30 probe, ddH2O4.8. mu.l, and the total volume of one reaction was 18. mu.l.
(3) Preparation of PCR amplification system for SMN1 and SMN2 gene copy number detection microdroplet digital PCR detection: add 18. mu.l of PCR reaction to 2. mu.l of DNA template and mix by gentle swirling.
(4) Droplet generation: the prepared 20 mul digital PCR amplification system is added into the water phase hole of the 8-channel micro-droplet generation chip, then 40 mul micro-droplet generation oil is added into the oil phase hole of the 8-channel micro-droplet generation chip, and the chip is placed in a MicroDrop-100A sample preparation instrument (Guangdong Yongno medical science and technology Co., Ltd.) to prepare PCR micro-reaction liquid drops.
(5) The prepared PCR micro-reaction droplets were carefully transferred to a 96-well reaction plate and heat-sealed with a sealing plate film.
(6) Putting the heat-sealed 96-pore plate into a PCR instrument, and setting a reaction program for PCR amplification, wherein the PCR amplification reaction program is as follows: 1 cycle at 95 ℃ for 10 min; 30 seconds of denaturation at 94 ℃, 60 seconds of annealing and extension at 58 ℃ for 45 cycles; 1 cycle at 98 ℃ for 10 min; maintaining at 16 ℃.
(7) After PCR amplification reaction, the 96-well plate is placed in a MicroDrop-100B biochip analyzer (Guangdong Yongnuo medical science and technology Co., Ltd.) to detect a fluorescent signal, a droplet signal detector with a nucleic acid molecule plate gives out the fluorescent signal, and a droplet signal detector without the template has no fluorescent signal. And finally, according to the Poisson distribution principle and the proportion of the positive microdroplets, calculating the copy number of the target molecules to be detected by using a micro drop multi-domain digital PCR instrument matched data analysis software Quantdrop.
EXAMPLE five
In this embodiment, the PCR reaction solution/kit described in example 3 is used to detect exon 7 of SMN1 and SMN2 genes, and the specific detection method is as follows:
(1) sample preparation: HT1080 cell line DNA was used at a concentration of 30 ng/. mu.l.
(2) The PCR reaction solution for detecting the copy number of the SMN1 and SMN2 genes by microdroplet digital PCR is prepared as follows: 10 μ L of Master Mix, 0.5 μ L of 20 μm/L forward primer, 0.5 μ L of 20 μm/L reverse primer, 0.6 μ L of 10 μm/L probe, ddH2O4.3. mu.l, restriction enzyme 0.5. mu.l, and the total volume of one reaction was 18. mu.l.
(3) Preparation of PCR amplification system for SMN1 and SMN2 gene copy number detection microdroplet digital PCR detection: add 18. mu.l of PCR reaction to 2. mu.l of DNA template and mix by gentle swirling.
(4) Droplet generation: the prepared 20 mul of digital PCR amplification system is added into the water phase hole of the 8-channel micro-droplet generation chip, then 40 mul of micro-droplet generation oil is added into the oil phase hole of the 8-channel micro-droplet generation chip, and the chip is placed in a MicroDrop-100A sample preparation instrument (Guangdong Yongno medical science and technology Co., Ltd.) to prepare PCR micro-reaction liquid drops.
(5) The prepared PCR micro-reaction droplets were carefully transferred to a 96-well reaction plate and heat-sealed with a sealing plate film.
(6) Putting the heat-sealed 96-pore plate into a PCR instrument, and setting a reaction program for PCR amplification, wherein the PCR amplification reaction program is as follows: 1 cycle at 95 ℃ for 10 min; performing denaturation at 95 ℃ for 30 seconds, annealing and extension at 58 ℃ for 60 seconds for 45 cycles; 1 cycle at 98 ℃ for 10 min; keeping at 4 ℃.
(7) After PCR amplification reaction, the 96-well plate is placed in a MicroDrop-100B biochip analyzer (Guangdong Yongnuo medical science and technology Co., Ltd.) to detect a fluorescent signal, a droplet signal detector with a nucleic acid molecule plate gives out the fluorescent signal, and a droplet signal detector without the template has no fluorescent signal. And finally, according to the Poisson distribution principle and the proportion of the positive microdroplets, calculating the copy number of the target molecules to be detected by using a micro drop multi-domain digital PCR instrument matched data analysis software Quantdrop.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention, and those skilled in the art may make modifications and variations within the spirit of the present invention, and all modifications, equivalents and modifications of the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.
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Claims (7)
1. A human SMN1 and SMN2 gene copy number detection kit based on a digital PCR method is characterized in that the kit comprises a DNA extraction reagent and a PCR reaction solution; wherein the PCR reaction solution comprises a primer for quantitative detection of human SMN1 and SMN2 genes, a primer for detection of an internal reference RPP30 gene, a human SMN1 and SMN2 gene detection probe and an internal reference RPP30 gene detection probe;
wherein, the nucleotide sequences of the primers for quantitative detection of the human SMN1 and SMN2 genes are shown as SEQ ID NO. 1 and SEQ ID NO. 2; the nucleotide sequence of the primer detected by the internal reference RPP30 gene is shown as SEQ ID NO. 3 and SEQ ID NO. 4;
the nucleotide sequences of the human SMN1 and SMN2 gene detection probes are shown as SEQ ID NO. 5; the nucleotide sequence of the internal reference RPP30 gene detection probe is shown in SEQ ID NO. 6.
2. The kit for detecting copy number of human SMN1 and SMN2 genes based on the digital PCR method as claimed in claim 1, wherein the human SMN1 and SMN2 gene detection probes are labeled with a fluorescent reporter at the 5 'end and a non-fluorescent quencher at the 3' end and connected with an MGB modifier.
3. The kit for detecting copy number of human SMN1 and SMN2 genes by digital PCR method according to claim 1, wherein the concentration of the primer for quantitative detection of human SMN1 and SMN2 genes and the concentration of the primer for detection of internal reference RPP30 gene are 10-50 μm/L respectively.
4. The kit for detecting copy number of human SMN1 and SMN2 genes by digital PCR method according to claim 1, wherein the concentrations of the human SMN1 and SMN2 gene detection probes and the reference RPP30 gene detection probe are 5-30 μm/L respectively.
5. The kit for detecting copy number of human SMN1 and SMN2 genes by digital PCR-based method according to claim 1, wherein the PCR reaction solution further comprises a PCR premix for microdroplet digital PCR.
6. The kit for detecting copy number of human SMN1 and SMN2 genes based on the digital PCR method as claimed in any one of claims 1 to 5, wherein the application of the kit comprises the following steps:
1) extracting DNA from a sample to be detected as a template;
2) preparing a PCR reaction solution;
3) preparing a PCR specific amplification reaction system;
4) generating microdroplets by using the PCR specific amplification reaction system;
5) performing digital PCR amplification;
6) the copy number of the target molecule to be detected is calculated by software.
7. The kit for detecting copy number of human SMN1 and SMN2 genes based on the digital PCR method as claimed in claim 6, wherein the PCR-specific amplification reaction system in the preparation 3) comprises: PCR premix, SMN1/2 forward primer, SMN1/2 reverse primer, SMN1/2 probe, RPP30 forward primer, RPP30 reverse primer, RPP30 probe, ddH2O and a DNA template.
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